A low molecular weight human, mouse and rat transforming growth factor (TGF) was isolated from serum-free medium conditioned by a human metastatic melanoma tumor line, a Moloney MuSV-transfored mouse 3T3 cell line, and a Synder-Theilen FeSV-transformed Fisher rat embryo fibroblast cell line. The estimated molecular weight of TGF is 7,400. It is a single chain polypeptide of 67 residues with three intrachain disulfide bridges. A comparison of the amino-terminal region of the first 26 residues of TGF from different species and different cell types shows 91% homology. Alignment of the amino acid sequence of TGF with the sequence of epidermal growth factor (EGF) reveals statistically significant sequence homology. TGF competes with I-125-EGF for the same receptor completely and equivalently. I-125-TGF did not discriminate between TGF and EGF. TGF stimulates, in the presence of A431 cell membranes, the phosphorylation of a synthetic polypeptide which corresponds to the sequence of the reported site of tyrosine phosphorylation in pp60-src. In contrast, TGF enabled normal anchorage-dependent cells to grow in soft agar, whereas EGF did not stimulate growth of these cells in semi-solid medium. Based on the chemical and biological data, it is proposed that TGF and EGF are chemically distinct, but share a common receptor binding site.